Coupling between Cytoplasmic Protein Phase Separation and Cholesterol-Rich Domains in the Plasma Membrane Facilitates T Cell Activation

Abstract

During T cell activation, phosphorylation of the transmembrane adaptor Linker of Activation of T-cells (LAT) leads to multivalent interactions with its effectors (Grb2 and Sos1) to form biomolecular condensates through protein phase separation. These condensates facilitate T cell activation by excluding inhibitory enzymes and proof-reading the transduction of activation signals. Intriguingly, LAT has also been widely observed to be enriched in ordered, cholesterol-driven lipid domains, suggesting the potential for coupling between membrane-associated protein condensates and lipid organization. We provide evidence for such coupling in both reconstituted model systems and in living T-cells. In vitro, we reconstituted condensates on phase-separating model membranes by conjugating the phosphorylated cytoplasmic domain of LAT to spin coated lipid multilayers. Conjugating LAT to saturated lipids led to condensate formation exclusively apposed to liquid ordered (Lo) regions, and vice versa, indicating strong coupling between lipid phase separation and the over-lying protein condensates. Formation of protein condensates on a homogeneous membrane templated membrane domain localization, with Lo phases forming exclusively below condensates. We observed similarly strong coupling between cytoplasmic protein condensates and micrometer-sized cholesterol domains in living cells. T-cells plated onto activating coverslips produced condensates enriched in LAT and Grb2, which were exclusively apposed to microscopic regions of cholesterol enrichment. These cholesterol-rich domains were T cell activation-dependent and overlapped with validated marker of lipid rafts (GPI-anchored proteins and ganglioside GM1). Further, raft-preferring transmembrane domains (TMDs) were recruited to the cholesterol-rich domains/protein condensates, whereas TMDs depleted from raft domains were excluded. Thus, we show that cytoplasmic LAT condensates induced by antigen stimulation of T-cells induce, are strongly coupled to, microscopic cholesterol-rich plasma membrane domains, facilitating T cell activation.